Method for tumor diagnosis comprising administering of palladium-substituted bacteriochlorophyll derivatives

ABSTRACT

Palladium-substituted Bacteriochlorophyll derivatives are administered to a subject suspected of having a tumor and the patient is then irradiated and the fluorescence of the suspected area is measured. A high fluorescence indicates a tumor site. A preferred derivative is Pd-Bacteriopheophorbide a.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional application of U.S.application Ser. No. 09/857,772, filed Jun. 11, 2001, which is thenational stage under 35 U.S.C. 371 of international applicationPCT/IL99/00673, filed Dec. 9, 1999 which designated the United States,and which international application was published under PCT Article21(2) in the English language.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[0002] The present invention concerns palladium-substitutedbacteriochlorophyll derivatives, processes and intermediates for theirpreparation and pharmaceutical compositions comprising the same as wellas their use in the field of in vivo photodynamic therapy and diagnosisand in vitro photodynamic killing of viruses and microorganisms.

Definitions And Abbreviations

[0003] BChl=bacteriochlorophyll a (Mg-containing 7, 8, 17,18-tetrahydroporphyrin having a phytyl or geranylgeranyl group atposition 17³, a COOCH₃ group at position 13², an H atom at position 13²,an acetyl group at position 3 and an ethyl group at position 8).

[0004] BChlide=bacteriochlorophyllide a (the C-17²-free carboxylic acidderived from BChl a).

[0005] BPhe=bacteriopheophytin a (BChl in which the central Mg atom isreplaced by two H atoms).

[0006] BPheid=bacteriopheophorbide a (the C-17²-free carboxylic acidderived from BPhe).

[0007] Pd-BPheid=Pd-bacteriopheophorbide a (the C-17²-free carboxylicacid derived from BPhe having a central Pd atom, a COOCH₃ group atposition 13², an H atom at position 13², an acetyl group at position 3and an ethyl group at position 8).

[0008] IUPAC numbering of the bacteriochlorophyll derivatives is usedthroughout the specification. Using this nomenclature, the naturalbacteriochlorophylls carry two carboxylic acid esters at positions 13²and 17², however they are esterified at positions 13³ and 17³.

[0009] There has been an increasing interest in the utilization ofphotosensitizers for cancer therapy. According to this technique, knownas photodynamic therapy (PDT), photosensitizers are applied for exampleto a tumor and the in situ photosentization produces compounds whichintoxicate the malignant cells.

[0010] Photodynamic therapy using porphyrins and related compounds has,by now, a fairly long history. Early work, in the 1940s, demonstratedthat porphyrin could be caused to fluoresce in irradiated tumor tissue.The porphyrins appeared to accumulate in these tissues, and were capableof absorbing light in situ, providing a means to detect the tumor by thelocation of the fluorescence. A widely used preparation in the earlystages of photodynamic treatment both for detection and for therapy wasa crude derivative of hematoporphyrin, also called hematoporphyrinderivative, HpD, or Lipson derivative prepared as described by Lipsonand coworkers in J Natl Cancer Inst (1961) 26:1-8. Considerable work hasbeen done using this preparation, and Dougherty and coworkers reportedthe use of this derivative in treatment of malignancy (Cancer Res (1978)38:2628-2635; J Natl Cancer Inst (1979) 62:231-237).

[0011] Dougherty and coworkers prepared a more effective form of thehematoporphyrin derivative which comprises a portion of HpD having anaggregate weight >10 kd. This form of the drug useful in photodynamictherapy is the subject of U.S. Pat. No. 4,649,151, is commerciallyavailable, and is in clinical trials.

[0012] The general principles of the use of light-absorbing compounds,especially those related to porphyrins, has been well established as atreatment for tumors when administered systematically. The differentialability of these preparations to destroy tumor, as opposed to normaltissue, is due to the homing effect of these preparations to theobjectionable cells. (See, for example, Dougherty, T. J., et al.,“Cancer: Principles and Practice of Oncology” (1982), V. T. de Vita,Jr., et al., eds. pp 1836-1844.). Efforts have been made to improve thehoming ability by conjugating hematoporphyrin derivative to antibodies.(See, for example, Mew, D., et al., J Immunol (1983) 130:1473-1477.).The mechanism of these drugs in killing cells seems to involve theformation of singlet oxygen upon irradiation (Weishaupt, K. R., et al.,Cancer Research 36:2326-232: (1976)).

[0013] The use of hematoporphyrin derivative or its active components inthe treatment of skin diseases using topical administration has alsobeen described in U.S. Pat. No. 4,753,958. In addition, the drugs havebeen used to sterilize biological samples containing infectiousorganisms such as bacteria and virus (Matthews, J. L., et al.,Transfusion 28:81-83 (1988)). Various other photosensitizing compoundshave also been used for this purpose, as set forth, for example, in U.S.Pat. No. 4,727,027.

[0014] In general, the methods to use radiation sensitizers of a varietyof structures to selectively impair the functioning of biologicalsubstrates both in vivo and in vitro are understood in the art. Thecompounds useful in these procedures must have a differential affinityfor the target biological substrate to be impaired or destroyed and mustbe capable of absorbing light so that the irradiated drug becomesactivated in a manner so as to have a deleterious effect on the adjacentcompositions and materials.

[0015] Because it is always desirable to optimize the performance oftherapeutics and diagnostics, variations on the porphyrin drugstraditionally used in treatment and diagnosis have been sought. A numberof general classes of photosensitizers have been suggested includingphthalocyanines, psoralen-related compounds, and multicyclic compoundswith resonant systems in general. Most similar to the compoundsdisclosed herein are various pheophorbide derivatives whose use inphotodynamic therapy has been described in EPO Application 220686 toNihon Metaphysics Company; ethylene diamine derivatives of pheophorbidefor this purpose described in Japanese Application J85/000981 to TamaSeikayaku, K. K., and Japanese Application J88/004805 which is directedto 10-Hydroxypheophorbide-a. In addition, Beems, E. M., et al., inPhotochemistry and Photobiology 46:639-643 (1987) disclose the use asphotosensitizers of two derivatives ofbacteriochlorophyll-a—bacteriochlorophyllin-a (also known asbacteriopheophorbide-a, which lacks the phytyl alcohol derivatized inbacteriochlorophyll-a) and bacteriochlorin-a (which lacks both thephytyl group and the Mg ion). These authors direct their attention tothese derivatives as being advantageous on the grounds of enhanced watersolubility as compared to bacteriochlorophyll-a.

[0016] EP 584552 and WO97/19081, both to Yeda Research and DevelopmentCo. Ltd., describe chlorophyll and bacteriochlorophyll derivatives andtheir use as PDT agents, and metaled bacteriochrophylls and theirpreparation by transmetalation of the corresponding Cd—BChl derivatives,respectively.

[0017] The problem remains to find suitable photosensitizers useful inphotodynamic therapy and diagnosis which are optimal for particulartargets and particular contexts. Thus, the invention provides anadditional group of photosensitizing compounds which becomes part of therepertoire of candidates for use in specific therapeutic and diagnosticsituations.

SUMMARY OF THE INVENTION

[0018] It has now been found, in accordance with the present invention,that the compounds of formula I, I′ or I″ below wherein A as definedbelow represents a substituent capable of allowing an efficient plasmatransfer and cell membrane penetration, are useful as PDT agents andpresent the advantages of enhanced solubility, stability and/orefficiency, compared with the known compounds.

[0019] The invention thus concerns the compounds of formula I, I′ or I″

[0020] wherein

[0021] A represents OH,

[0022] OR₁,

[0023] —O—(CH₂)_(n)—Y,

[0024] —S—(CH₂)_(n)—Y,

[0025] —NH—(CH₂)_(n)—Y,

[0026] —O—(CH₂)₂—NH₂,

[0027] —O—(CH₂)₂—OH,

[0028] —NH—(CH₂)_(n)—⁺N O, X⁻,

[0029] —NH—(CH₂)₂—NH—BOC or

[0030] —N—(CH₂—CH═CH₂)₂

[0031] wherein

[0032] R₁ represents Na⁺, K⁺, (Ca²⁺)_(0.5), (Mg²⁺)_(0.5), Li⁺, NH₄⁺⁺NH₃—C (CH₂OH)₃, ⁺NH₃—CH₂—(CHOH)₄—CH₂OH, ⁺NH₂(CH₃)—CH₂—(CHOH)₄—CH₂OH or⁺N(C_(n), H_(2n),₊₁)₄;

[0033] R₂ represents H, OH or COOR₄, wherein R₄ is C₁-C₁₂ alkyl orC₃-C₁₂ cycloalkyl;

[0034] R₃ represents H, OH or C₁-C₁₂ alkyl or alkoxy;

[0035] n is 1, 2, 3, 4, 5 or 6,

[0036] Y is —NR′₁R′₂ or —⁺NR′₁R′₂R′₃, X⁻ wherein R′₁, R′₂ and R′₃independently from each other represent —CH₃ or —C₂H₅;

[0037] X is F, Cl, Br or I,

[0038] n′ is 1, 2, 3 or 4,

[0039] and wherein * denotes an asymmetric carbon and—represents asingle saturated bond or a double unsaturated bond.

[0040] Furthermore, the present invention concerns processes for thepreparation of the above new compounds.

[0041] Thus, in one aspect, it is herein described a method to effectthe impairment or destruction of a target biological substrate whichmethod comprises treating the target substrate with an amount of thecompound of formula I, I′ or I″ effective to photosensitive saidsubstrate followed by irradiating said target substrate with radiationin a wavelength band absorbed by the compound of formula I, I′ or I″ fora time effective to impair or destroy the substrate.

[0042] In other aspect, the invention is therefore directed topharmaceutical compositions comprising at least a compound of formula I,I′ or I″ as an active agent, together with a pharmaceutically acceptablecarrier. The compositions are useful for in vivo photodynamic therapyand diagnosis of tumors and for killing of cells, viruses and bacteria,parasites and fungi in samples and in living tissues by well-knownphotodynamic techniques.

[0043] Furthermore, the invention concerns the use of the compounds offormula I, I′ or I″ for the preparation of a pharmaceutical compositionuseful in photodynamic therapy.

[0044] The invention further concerns the use of the invention compoundsfor the preparation of compositions useful in diagnosis and ex vivokilling of bacteria, parasites, viruses and fungi.

[0045] The invention further concerns the acid chloride and anhydride offormulas II and III herebelow, respectively, as intermediates.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046]FIG. 1 depicts the optical absorption spectrum of Pd-BPheid in amixture of acetone and methanol/K buffer phosphate.

[0047]FIGS. 2 and 3 depict, respectively, low and high resolution massspectra of Pd-BPheid conducted by Fast Atom Bombardement (FAB-MS).

[0048]FIG. 4 shows time-dependent morphological changes of A431 cellswith Pd-BPheid or BChl-SerOMe post PDT. (In the Figures, Bchl-Ser standsfor BChl-SerOMe, the seryl methyl ester of BChl.)

[0049]FIG. 5 shows phototoxicity of Pd-BPheid and BChl-SerOMe tested onECV-304 cells.

[0050]FIG. 6 shows phototoxicity of Pd-BPheid and Pd-BPheid-ethyl esteron cultured M2R mouse melanoma cells. (A) pigments dissolved in 95%ethanol and further diluted to the indicated concentrations in culturemedium+10% serum to 1% ethanol. (B) pigments dissolved directly inculture medium+10% serum.

[0051]FIG. 7 shows phototoxicity of Pd-BPheid on cultured M2R mousemelanoma and human H29 colon carcinoma cells.

[0052]FIG. 8 shows PDT of M2R mouse melanoma with Pd-BPheid (2.5 mg/Kg)dissolved in Cremophor and diluted in salt solution.

[0053]FIG. 9 shows PDT of M2R mouse melanoma with Pd-BPheid (2.5 mg/Kg)dissolved in salt solution and diluted with Cremophor.

[0054]FIG. 10 illustrates cure of primary C6 glioma tumors after PDTwith Pd-BPheid or Pd-BPheid-SerOMe (in the Figure, Pd-Bchl-Ser).

[0055]FIG. 11 shows appearance of C6 glioma metastases in CD1 nude miceafter surgery (amputation) or after PDT with Pd-BPheid orPd-BPheid-SerOMe (in the Figure, Pd-Bchl-Ser).

DETAILED DESCRIPTION OF THE INVENTION

[0056] In a preferred embodiment, the compounds of the invention havethe following formula with the optical configuration below:

[0057] wherein A is as above.

[0058] When the dotted lines representing the bond between C7 and C8 andC17 and C18 in the above structure is a saturated single bond, thecarbon atoms numbered 7, 8, 17 and 18 are asymmetric carbon atoms. WhenR₂ or R₃ is H, C13² is an asymmetric carbon atom.

[0059] In the presence of oxygen or at the ambient air and under lightaction, the oxidation of the above C7-C8 and C17-C18 bonds may occur,resulting in compounds with double bonds at said positions C7-C8 andC17-C18.

[0060] The compounds of formula I′ and I″ of the present invention areoxidized forms of the compounds of formula I and can be obtained by theprocesses described in Chlorophyll, by Scheer H. (ed.), CRC Press, 1991,pp. 147-209.

[0061] In a preferred embodiment of the invention, the compounds arethose wherein A is OR₁.

[0062] In a most preferred embodiment, the compound of the invention isPd-PBheid (also designated herein sometimes Pd—BChl—COOH), the compoundof formula I wherein A is OH, having the following structure:

[0063] One of the processes for the preparation of the compounds offormula I wherein A is OH, comprises at least the steps of:

[0064] a) combined demetalation and hydrolysis of a M-BPheid-17³-Zcompound wherein Z is phytyl, geranylgeranyl (gg) or SerOMe (serylO-methyl ester) and M is a metal selected from Mg, Cd, or Zn;

[0065] b) incorporation of Pd with a Pd reagent into the compoundobtained in (a), thus obtaining a Pd-BPheid, and, if desired,

[0066] c) subsequent reaction of the obtained Pd-BPheid with acorresponding compound of formula A-H, wheren A is other than OH forforming the corresponding R₁ salt or a compound wherein A is not OH.

[0067] In one preferred embodiment, the process is directed to thepreparation of Pd-BPheid and bacteriochlorophyll a (Bchla) isdemetalated and hydrolyzed in step (a), and the obtainedbacteriopheophorbide (BPheid) is reacted with a Pd reagent in step (b)to produce the desired Pd-BPheid.

[0068] Another process for the preparation of the compound of formula Icomprises at least the steps of:

[0069] a) transmetalation of a BChlide-17³-Z to obtain the correspondingPd- BPheid-17³-Z wherein Z is phytyl, gg or Ser OMe,

[0070] b) hydrolysis of the obtained compound, and

[0071] c) optionally subsequent reaction of the obtained Pd-BPheid witha corresponding compound of formula R₁—H or A—H for forming thecorresponding R₁ salt or a compound wherein A is not OH.

[0072] In one preferred embodiment, the process is directed to thepreparation of Pd-BPheid and bacteriochlorophyll a (Bchla) istransmetalated in step (a) to replace the native central Mg atom by Pd,and the obtained Pd-BPheid-17³-Z wherein Z is phytyl is hydrolized instep (b) to produce the desired Pd-BPheid.

[0073] Another process for the preparation of the compound of formula Icomprises at least the steps of:

[0074] a) enzymatic hydrolysis of a BChlide-17³-Z wherein Z is phytyl orgeranylgeranyl to obtain a Bchlide;

[0075] b) acidic demetalation of said BChlide of (a);

[0076] c) incorporation of Pd with a Pd reagent into the demetalatedBPheid of (b); and

[0077] d) optionally subsequent reaction of the obtained Pd-BPheid witha corresponding compound of formula A—H, wherein A is other than OH, forforming the corresponding R₁ salt or a compound wherein A is not OH.

[0078] In the above processes for the preparation of compounds offormula I, the Pd reagent may be any convenient reactive compoundproviding Pd in such structures such as, for instance, Pd acetate and Pdchloride.

[0079] The incorporation of Pd in the procedures above can be achievedby a two-step procedure using Na ascorbate or ascorbic acid, or by aone-step procedure using 6-O-palmitoyl-L-ascorbic acid.

[0080] The compounds of the invention wherein A is different from OH andOR₁ may be obtained by reaction of the Pd-BPheid (Pd—BChl—COOH) with thecorresponding A—H compound.

[0081] The compounds of formula II and III above are intermediates forthe compounds of formula I of the invention. The acid chlorides offormula II, Pd-BPheid-COCl, may be obtained by using any agent suitablefor forming acyl chlorides, such as for example SOCl₂.

[0082] The acid anhydrides of formula III may be obtained by dehydrationof the compounds of formula I, I′, I″ with acetic anhydride.

[0083] By reaction of these intermediates II and III with thecorresponding compound AH, the compounds of formula I, I′ or I″ may beobtained.

[0084] The invention further comprises pharmaceutically acceptable saltsof the free acids of formulas I, I′ and I″. The salts can be formed bymethods well known in the art such as by reaction of the free acid or asalt thereof with inorganic or organic reagents such as, but not limitedto, NaOH, KOH, calcium or magnesium suitable salts, LiOH, NH₄OH,tetraalkylammonium hydroxide, e.g., tetraethylammonium hydroxide, orN-methylglucamine, glucamine and triethanolamine.

[0085] The compounds of the invention are for use in photodynamictherapy and diagnosis with respect to target biological substrates. By“target biological substrate” is meant any cells, viruses or tissueswhich are undesirable in the environment to which therapy or othercorrective action, such as sterilization, is employed, or the locationof which is desired to be known in an environment to which diagnosis isapplied.

[0086] According to the present invention, the drug is injected into thesubject, and permitted to reach an optimal concentration in the targetsubstrate. Then the target substrate is exposed to radiation at awavelength appropriate to the absorption spectrum of the compoundadministered. The effect of the compound can be enhanced by concomitantincrease of the target substrate temperature.

[0087] For use in the method of the invention, the compounds of theinvention are formulated using conventional excipients appropriate forthe intended use. For systemic administration, in general, bufferedaqueous compositions are employed, with sufficient nontoxic detergent tosolubilize the active compound. As the compounds of the invention aregenerally not very soluble in water, a solubilizing amount of suchdetergent may be employed. Suitable nontoxic detergents include, but arenot limited to, Tween-80, various bile salts, such as sodium glycholate,various bile salt analogs such as the fusidates. Alternate compositionsutilize liposome carriers. The solution is buffered at a desirable pHusing conventional buffers such as Hank's solution, Ringer's solution,or phosphate buffer. Other components which do not interfere with theactivity of the drug may also be included, such as stabilizing amountsof proteins, for example, serum albumin, or low density- or highdensity-lipoprotein (LDL and HDL, respectively).

[0088] Systemic formulations can be administered by injection, such asintravenous (i.v.), intraperitoneal (i.p.), intramuscular, orsubcutaneous (s.c.) injection, or can be administered by transmembraneor transdermal techniques. Formulations appropriate for transdermal ortransmembrane administration include sprays and suppositories containingpenetrants, which can often be the detergents described above.

[0089] For topical local administration, the formulation may alsocontain a penetrant and is in the form of an ointment, salve, liniment,cream, or oil. Suitable formulations for both systemic and localizedtopical administration are found in Remington's Pharmaceutical Sciences,latest edition, Mack Publishing Co., Easton, Pa.

[0090] For use ex vivo to treat, for example, blood or plasma fortransfusion or preparations of blood products, no special formulation isnecessary, but the compounds of the invention are dissolved in asuitable compatible solvent and mixed into the biological fluid at asuitable concentration, typically of the order of 1-100 μg/ml prior toirradiation.

[0091] For photodynamic therapeutic and diagnostic applications,suitable dosage ranges will vary with the mode of application and thechoice of the compound, as well as the nature of the condition beingtreated or diagnosed. However, in general, suitable dosages are of theorder of 0,01 to 50 mg/kg body weight, preferably 0,1 to 10 mg/kg. Fortopical administration, typically amounts on the order of 5-100 mg totalare employed.

[0092] The general procedures for photodynamic ex vivo treatment areanalogous to those described by Matthews, J. L., et al., Transfusion(supra).

[0093] Briefly, for systemic administration, a suitable time periodafter administration, typically from several minutes to two days isallowed to elapse in order to permit optimal concentration of thecompounds of the invention in the target biological substrate. Ingeneral, this substrate will be a tumor vasculature, tumor cells or anyother tumor component, and the localization of the compound can bemonitored by measuring the optical absorption of the target tissue ascompared to background. After optimization has been accomplished, thetarget biological substrate is irradiated with a suitable band ofirradiation, in the range of 740-800 nm, or 500-600 nm or 700-900 nm ata rate of 5-750 mW/cm², and a total energy of 100-1000 J/cm².

[0094] For topical treatment, localization is immediate, and thecorresponding radiation can be provided thereafter. For treatment ofbiological fluids ex vivo, radiation is applied after optimalbinding/uptake by the target tissue is reached. The radiation fluence ison the order of 1-10 J/cm². Because penetration of tissue is notrequired, lower total energy can be employed.

[0095] The compositions of the invention comprise at least one compoundof formula I, I′ or I″ as defined above together with a physiologicallyacceptable carrier. These compositions may be in the form of a solution,a lipid emulsion or a gel or in the form of liposomes or nanoparticles.The suitable carrier is chosen to allow optimization of theconcentration of the compound of the invention at the target substrate.Examples of such carriers, but not limited to, are “Tween 80”,polyethyleneglycol, e.g., PEG400, “Cremophor EL”, propylene glycol,ethanol, basil oil, bile salts and bile salts analogs and mixturesthereof. Liposome formulations can be based, for example, ondimyristoylphosphatidyl choline or phosphatidyl glycerol. The carriermay also comprise dipalmitoylphosphatidyl choline.

[0096] When nanoparticles are used, they may be in the form ofPEG-coated poly(lactic acid) nanoparticles. In the form of lipidemulsions, low density lipoproteins and triglycerides are usually used.

[0097] In the composition of the invention, the invention compound(s) is(are) in an amount of 0.01 to 20%, preferably 0.05% to 5% by weight ofthe total weight composition.

[0098] The invention will now be illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Preparation of Pd-BPheid

[0099] Pd-BPheid was prepared from BChla by the following 3-stepprocedure.

[0100] (a) Isolation of Bacteriochlorophyll a (BChla)

[0101] BChla was extracted form lyophilized bacteria Rhodovolumsulfidophilum as follows:

[0102] Lyophilized cells (100 gr) were ground to powder, washed 5 timeswith a total of 1250 ml acetone to partially wash away the carotenoids,the mixture was filtered and BChla was extracted from the solid withabsolute methanol (≈1200 ml, 4-5 filtrations). After filtering, the darkblue-green solution was partly evaporated under vacuum, the concentratedsolution (≈500 ml) was extracted 2-3 times with petrol ether (b.p.80-100° C., ≈1300 ml) to further eliminate carotenoids, and the petrolether phase was extracted twice with methanol (≈550 ml). This phase wasthen discarded, the combined methanol phase was evaporated under vacuum,and the blue-green residue was redissolved in methanol-acetone (1:3,v/v) and loaded on a DEAE-Sepharose column (3×10 cm) equilibrated withmethanol-acetone (1:3, v/v). The BChla was eluted with methanol-acetone(1:3, v/v), the methanol-acetone mixture was evaporated and the dryBchla was redissolved in an exact volume (for absorption spectrum) ofether and filtered through cotton wool to get rid of dissolved columnmaterial. After a final evaporation the solid pigment was stored underArgon in the dark at −20° C. Extraction yield: about 700 mg BChla per100 g lyophilized cells.

[0103] The DEAE-Sepharose column was prepared as previously described(Omata and Murata, 1983, “Preparation of Chlorophyll a, Chlorophyll band Bacteriochlorophyll a by column chromatography with DEAE-SepharoseC1-6B and Sepharose C1-6B”, Plant Cell Physiol 24:1093-1100). Briefly,DEAE-Sepharose was washed with distilled water and then converted to anacetate form by suspending it in a 1M sodium acetate buffer (pH=7). Theslurry was washed 3 times with acetone and finally suspended inmethanol-acetone (1:3, v:v) for storage at 5° C.

[0104] (b) Preparation of Bacteriopheophorbide (BPheid)

[0105] Crude Bchla extract as obtained in (a) (about 100 mg Bchlacontaining some residual carotenoides) was dissolved in 80% aqueoustrifluoroacetic acid (about 15 ml) which had been bubbled with nitrogenfor 10 min. The solution was stirred at ambient temperature for 2 h.Then the reaction mixture was poured into water (250 ml) and extractedwith chloroform. The extract was washed twice with water and dried overanhydrous Na₂SO₄. After evaporation of the solvent the residue waschromatographed on Silica (3 cm×15 cm column, Kieselgel 60, Merck) andeluted with methanol in chloroform by step gradient: 2%, 5%, 10%, 15%.At the beginning, carotenoids and a small amount of bacteriopheophytinwere washed out, followed by elution of allo-bacteriopheophytin andcarotenoids. At 10% methanol in chloroform the product started to becollected and monitored by TLC (Kieselgel, chloroform-methanol, 9:1).The product (60 mg) was evaporated, and the residue taken up in CHCl₃was filtered through UltraPore membrane to remove residual silica thatcould otherwise cause oxidations.

[0106] (c) Incorporation of Palladium into Bacteriopheophorbide (Bpheid)

[0107] BPheid (100 mg) as obtained in (b) and Pd-acetate (80 mg) weredissolved in dichloromethane (≈10 ml) and added to a suspension of 200mg sodium ascorbate in 50 ml of methanol. The reaction mixture wasstirred in a closed flask at room temperature, and samples from thereaction mixture were collected every 15-20 minutes and their opticalabsorption recorded. After about 4 hours, most of the BPheid absorptionat 357 nm was replaced by the Pd-BPheid absorption at 330 and 390 nm.

[0108] The reaction mixture was transferred into a chlorofom/watersolution (200 ml; 50:50 v/v) and shaken in a separatory funnel. Theorganic phase was collected, washed with water, dried over anhydroussodium chloride, and evaporated. The dried material was added to 80 mgof Pd-Acetate and steps above were repeated until the residualabsorption at 357 nm completely vanished and the ratio between theabsorption at 765 nm (the peak of the red-most transition) and theabsorption maximum at 330 nm reached the value of ≈2.4 (in chloroform).

[0109] The dried reaction mixture was solubilized in a minimal volume of2:1 chloroform:acetone and loaded on a CM-Sepharose column (150 mm×25mm) that had been pre-equilibrated with acetone. The column was firstwashed with acetone and the eluted first fraction was discarded. Thecolumn was then washed with 9:1 acetone:methanol. Two bands becameprominent and were washed out—the first was the major product and thesecond was an allomerized by-product (discarded). The product wasconcentrated almost to dryness and transferred into a 50:50chlorophorm:water system in a separatory funnel. The mixture wasthoroughly shaken and the organic phase was separated, dried overanhydrous sodium sulfate (or sodium chloride) and evaporated to dryness.

Example 2 Preparation of Pd-BPheid

[0110] (a) Isolation of Bchla

[0111] This step of the procedure was carried out as in Example 1(a)above.

[0112] (b) Preparation of Pd-Bpheid

[0113] 6-O-palmitoyl-L-ascorbic acid (246 mg, 593 μmol) was dissolved inMeOH (84 ml) and N₂ was passed through the solution. Bpheid (92 mg, 151μmol) and Pd(CH₃COO)₂ (83 mg, 370 μmol) were dissolved in CHCl₃ (34 ml,degassed with N₂) and added to the methanolic solution. The mixture waskept under inert atmosphere by stirring and the reaction progress wasmonitored by recording the absorption spectra of small reaction portionsevery few minutes. After ˜30 min. the reaction was completed and thesolvents were evaporated.

[0114] (c) Purification of Pd-Bpheid

[0115] The crude Pd-BPheid was dissolved in CHCl₃ and loaded on a columnpacked with 15 g of 0.4%-Silica-Asc. Small volume of CHCl₃ (˜30 m)l waspassed through the column and than the pigment was eluted usingMeOH:CHCl₃ (1:99, ˜250 ml). Purity of the fractions was determined byTLC and optical absorption spectroscopy. Mass Spectroscopy and NMRdetection were performed on representative samples. Yield: 82.5 mg ofpure Pd-BPheid (76%).

[0116] For the preparation of the 0.4%-Silica-Asc, ascorbic acid (240mg) was dissolved in 240 cc of EtOH:CHCl₃:MeOH (60:60:120) mixture.Silica gel 60 (60 g, Merck, Cat. No. 107734, mesh 70-230) was added andthe slurry mixture was stirred for 10 min. and then filtered at thepump. The yellowish Silica-Asc. was finally dried for ˜1 hr. at ˜50° C.This 0.4%-Silica-Asc. is ready to use as regular silica gel; its natureis less polar and it has some antioxidative properties.

Example 3 Preparation of Pd-BPheid

[0117] (a) Isolation of Bchla

[0118] This step was performed as in Example 1(a) above.

[0119] (b) Preparation of Chlorophyllase (Chlase)

[0120] Chlorophyllase (Chlase) was prepared from chloroplasts of Meliaazedarach L., Chine tree leafs. Fresh leaves (50 g) were ground for 2min. in a blender containing 350 ml of acetone cooled to −20° C. Thehomogenate was filtered through four layers of gauze, and the filtratewas collected and left overnight at 4° C. for further precipitation. Theacetone was removed by filtration, and the remaining powder was washed afew times with cold acetone to remove traces of Chlase and carotenoidsuntil the filtrate was colorless. The Chlase acetone powder was finallydried in a lyophilizer and further stored at −20° C. Under theseconditions, the enzyme preparation was stable for over 1 year withoutnoticeable loss of activity. Yield: 20 g Chlase per 1 kg leaves wereobtained.

[0121] (c) Synthesis and Purification of Bacteriochlorophyllide(BChlide)

[0122] Ascorbic acid (70 mg; Merck) was dissolved in water (9 ml), thepH of the solution was adjusted to 7.7 using 10 M KOH aqueous solution,and 1 ml of 0.5 M sodium phosphate buffer (pH 7.7) was added to maintainthe pH during the reaction. Triton X-100 (about 80 μl) was added toachieve a final detergent concentration of 0.8% (v/v). Chlase acetonepowder (200 mg) was homogenized in 6 ml of this solution using aPolytron homogenizer. The remaining solution was used to wash theinstrument and was then combined with the homogenate. The enzymesolution was sonicated with 20 mg of solid BChla saturated with Argonand incubated in the dark for 6 hrs at 37° C., while stirring.

[0123] For purification, the reaction material was directly frozen (−20°C.) after 6 hrs of reaction and subsequently lyophilized. The dryresidue was dissolved in acetone and sonicated and the solution was thensubjected to a CM-Sepharose column equilibrated in acetone. The columnwas washed with acetone to elute unreacted material and then with 5% and7% methanol (v/v) in acetone to elute Bacteriochlorophyllide (Bchlide)and Bacteriopheophorbide (Bpheid). The product was eluted with 25%methanol in acetone. The solvent was evaporated and the solid pigmentwas stored under Argon, at −20° C., in the dark. Reaction yield: 30-55%.

[0124] The CM-Sepharose for chromatography was prepared by first washingCM-Sepharose with water and then 3 times with acetone before packing acolumn and equilibrating in acetone. The chromatographic material couldbe reused after thorough rinsing with 2M NaCl aqueous solution untilcolorless, washed with water and resuspended in acetone.

[0125] (d) Incorporation of Palladium into the Bacteriopheophorbide(BPheid)

[0126] The procedure is the same as in Example 1 (c) above. HPLC of thedried material showed the main product in the form of two epimers whichwere chemically identical (88% of the entire mixture) and residualallomers. There was also a slight (0.5%) contamination of the startingmaterial, BPheid.

Example 4 Characterization of the Compound Pd-BPheid

[0127] (a) Absorbance Spectra

[0128] The absorbance spectra of Pd-BPheid were determined with a UVICONspectrophotometer (1 cm pathlength) using a PM detector which isnormalized to baseline. The sensitivity is 0.05.

[0129] Absorbance spectra of Pd-Bpheid in acetone and a mixture ofmethanol/K phosphate buffer are reported in Table 1 and in FIG. 1.

[0130] The absorbance spectrum of Pd-BPheid in plasma was red-shifted to763 nm. TABLE 1 Methanol/K Phosphate 20 mM pH 6.59 Acetone (70% / 30%) λAbsorbance λ Absorbance 753 nm 2.43 758 nm 1.25  530 nm 0.49 537 nm0.324 385 nm 1.25 384 nm 0.535 331 nm 1.45 329 nm 0.777

[0131] The pic detection revealed the following peaks according to FIG.1: at 758 nm: 1.2502; at 537 nm: 0.3239; at 384 nm: 0.5351; and at 329nm: 0.7766.

[0132] (b) HPLC Detection of Pd-BPheid

[0133] A reverse phase HPLC method was developed to characterize theimpurity profile and quantify the Palladium-BPheid. Solid phase a C₈Inertsil 5 μm, 250 × 4.6 mm Liquid phase methanol:potassium phosphatebuffer 20 mM pH = 6.59 (70%:30%) Flow rate 1 ml/min Volume of injection100 μl Detection 1-Spectroflow 783, Deuterium lamp: 385 nm 2-Spectroflow757, Tungsten lamp: 753 nm

[0134] As shown in Table 2, HPLC analysis of the product Pd-Bpheid asobtained in Example 3 exhibited 7 peaks. The major peak represented 64to 70% of the total products.

[0135] Solutions of Pd-BPheid stored in acetone at −20° C. were stablefor at least 2-month period. When the stock solution was maintained atroom temperature for 18 hours, no change in the HPLC profile wasobserved showing that Pd-Bpheid is a stable compound. TABLE 2 HPLCDetection of Pd-BPheid Absorption % % spectra (wavelength Peak Detection385 nm Detection 753 nm of maxima nm) B1 0.7 0.78 B2 3.4 4.31754,537,384,330 C 1.07 1.25 D 2.49 2.76 758,535,384,330 E 64.11 69.98758,537,384,329 F 9.62 3.61    753,531,358 G 13.56 14.46 758,537,384,329

[0136] (c) Characterization of Pd-BPheid by NMR

[0137] After a purification step of the Pd-BPheid prepared according tothe Example 3, the percentage of the major peak was above 90%. Thispurification was conducted by a preparative HPLC C8. This purifiedcompound was used for the characterization of the product by NMR andmass spectrometry.

[0138] Analysis of Pd-BPheid by NMR was carried out and the chemicalshifts are listed in Table 3:

[0139]¹H NMR and ¹³C NMR

[0140] 2D¹H NMR (COSY and NOESY)

[0141] 2D¹H-¹³C NMR (HMQC and HMBC: reverse detection). TABLE 3 ¹H¹³CChemical Shifts (ppm) Methyl Proton Carbon  1-CH₃ 3,44 14,4  2-CH₃ 3,0733,1  3-CH₃ 1,75 23,6  4-CH₃ 1,06 10,8  5-CH₃ 3,36 12,5  8-CH₃ 1,65 23,910-CH₃ 3,85 53,3 Meso α 9,11 101,5  β 8,50 102,9  δ 8,45 98,7 C—H  3-H4,35 47,2  4-H 4,09 55,2  7-H 4,10 49,2  8-H 4,34 49,2 10-H 5,92  65,10Others  4-CH₂ 2,08; 2,22    30,6*  7′-CH₂ 2,30; 2,52    30,6*  7″-CH₂2,15; 2,35 35* Carbon without Proton  2-CO 199  9-CO 188 17-CO₂H 170,2 10-CO₂Me 174,3   1-C 141  2-C 135,6   5-C 126,9   6-C 130,1  11-C 142,3 12-C 158,5  13-C 159,5  14-C 151,5  15-C 140,5  16-C 152,5  17-C 109,8 18-C 152,3  19-C 158,6 

[0142] (d) Characterization of Pd-BPheid by Mass Spectrometry

[0143] The mass spectrometry analysis of Pd-BPheid resulted in thespectra depicted in FIGS. 2 and 3. It was conducted by Fast AtomBombardment (FAB) under low and high resolutions. The spectrometer was a“ZabSpec TOF Micromass” spectrometer; ionisation mod: LSIMS with Cs⁺,positive, acceleration: 8 kV; source temperature: 40° C.; solvent used:mNBA (meta-nitrobenzilic alcohol); input: lateral.

[0144] Results: iontype: M+; formula: C₃₅H₃₆N₄O₆ ¹⁰⁶Pd; theory: 714.1670Z:1m/z theoretical 714.1670 m/z found 714.1689.

[0145] These results confirmed the NMR study: m/e=714 and confirmed theinsertion of Palladium metal.

[0146] The chemical structure analyzed by NMR and mass spectrometry isthe palladium derivative of the free acid form of BChl-Pd-BPheid.

Example 5 Biological Activity of Pd-Bpheid on Murine L1210 and Humanht29 Cells

[0147] (i) Cell Lines.

[0148] The murine leukemia cell line (L1210) was maintained insuspension culture using Fischer's medium supplemented with 10% horseserum, 1 mM glutamine, 1 mM mercaptoethanol and gentamicin. The RIF(Radiation induced Fibrosarcoma) tumor was maintained as specified byTwentyman et al. (1980, “A new mouse tumor model system (RIF-1) forcomparison of end-point studies”, J Natl Cancer Inst 64:595-604).Cultures were grown in Weymouth's medium containing 10% fetal calf serumand gentamycin.

[0149] HT29 human colon adenocarcinoma cells were cultured in RPMI 1640without phenol red and with 10% FCS. Cells were subcultured by dispersalwith 0.25% trypsin in 0.02% EDTA and replated at a 1:5 split.

[0150] (ii) In Vitro Phototoxicity.

[0151] For studies on phototoxicity involving L1210 and RIF cells, lightwas provided by a 600 watt quartz-halogen source filtered with 10 cm ofwater and a 850 nm cut-off filter to remove IR. The bandwidth wasfurther confined to 660±5 nm by an interference filter (Oriel). Cells insuspension (L1210) or adhering to 24 mm diameter cover slips wereincubated in growth medium (with 20 mM HEPES pH 7 replacing NaHCO₃ foradded buffering capacity) for 15 min in the presence of specified levelsof sensitizers. The cells were then washed free from the sensitizer, andtransferred to fresh media. Irradiations were carried out at 10° C. Forsome studies, the cells were then labeled with fluorescent probes andsites of photodamage were assessed. In other studies, the cells werethen incubated for 60 min at 37° C. in fresh medium to allow apoptosisto proceed. Viability studies were carried out using 96-well plates anda 72-hour MTT assay, in quadruplicate.

[0152] For HT29 model, cells were incubated for 1 hour with differentconcentrations of Pd-Bpheid and irradiated by an halogen lamp or atitanium sapphire laser with 300 mW/cm² at 10 and 25 J/cm².

[0153] (iii) Cell Viability.

[0154] Cell survival was assessed by the MTT reaction carried out 3 daysafter plating of 1,000-50,000 cells in 96 well plates. The colorintensity was compared to a standard curve containing variable numbersof control cells. Absorbance at x nm was determined with a BioRad Platereader. For L1210, growth in fresh medium was allowed to occur duringthe next 3 days, and cell numbers were similarly estimated using the MTTassay procedure.

[0155] (iv) Lipoprotein Binding.

[0156] Binding of Pd-BPheid to protein and lipoprotein compound ofcontrol human plasma was determined. Incubation of 250 μl plasma samplewith 3 μM of the compound for 30 min at 37° C. Lipoprotein and proteincomponents were then separated by density-gradient centrifugator. Thegradients were fractionated, fractions diluted into 3 ml of 10 mM TritonX-100 detergent or of fluorescence at 750-800 nm determined uponexcitation at 400 nm.

[0157] Results

[0158] (v) Phototoxicity Effect of Pd-BPheid on L1210 Cells.

[0159] L1210 murine leukemia cells were incubated with 1 μM Pd-BPheidfor 30 min at 37° C. resulting in a 50% cell killing using a 75 mJ/cm²dose of light at 760±5 nm. A similar degree of cell killing in the RIFline required a 215 mJ/cm² light dose.

[0160] (vi) Phototoxicity Effect of Pd-BPheid on HT29 Cells.

[0161] The survival rate varied between 100% and 79% when HT29 cellswere incubated with Pd-BPheid without light. The cellular survival ratedecreased when the concentration of Pd-BPheid was higher and when thedoses of energy delivered were increased. The Pd-BPheid photosensitizerdose causing a 50% death rate (also called LD₅₀) was 48 μM under anirradiation of 25 J/cm². The excitation wavelength inducing the mostimportant phototoxicity was 773 nm.

[0162] (vii) Sites of Photodamage.

[0163] Using mouse leukemia L1210 cells, Pd BPheid was highly specificmitochondrial photosensitizers with no detectable photodamage to theplasma membrane or to lysosomes. Such a result has been associated withrapid initiation of apoptosis.

[0164] (viii) Plasma Lipoprotein Binding.

[0165] Studies carried out indicated that Pd-BPheid bound to HDL>LDL>>>Albumin fractions of human serum, considered to be one determinant ofPDT selectivity.

Example 6 Formulations of Pd-Bpheid: Solubilization and Stability ofPd-Bpheid in Solvents Used for Animal Experiments

[0166] Solutions of Pd-BPheid were made up in different formulations toobtain a concentration of 0.05 to 2%.

[0167] (a) Cremophor formulation was prepared as follows: 40 mg ofPd-BPheid was dissolved in 2 ml of Cremophor EL in a dry tube either byslow rotation of the vial until the solution had been completely freefrom particles, or using short pulses of a sonic oscillator probe. Thetube was cooled such that temperature did not rise above 30° C. Afterthe drug was solubilized, 0.6 ml of propylene glycol was added and againmixed either by slow rotation or with the sonic probe. Isotonic NaCl wasthen added in 0.1 ml portions to a total volume of 4 ml. The mixtureshould be clear after each addition, with no evidence of a precipitate.The compositions were briefly treated with the sonic probe after eachaddition of NaCl 0.9% taking care to keep the temperature below 25-30°C. The concentration of drug was assessed by measuring the absorbance at757 nm after dilution into ethanol.

[0168] When 20 mg/kg of Pd-BPheid were used in experimental studies,this translated into 0.4 mg per 20 gram mouse. Since no more than 0.1 mlof Cremophor can be injected into a tail vein, the drug concentrationwas then 4 mg/ml.

[0169] (b) A modified Cremophor formulation was prepared as follows: 5mg of Pd-BPheid was mixed with 0.4 ml of Cremophor EL. Afterdissolution, 0.12 ml of propylene glycol was added. Isotonic saline(1.48 ml) was then added in small portions, and the same was mixed aftereach addition. The final solution was completely clear and free fromparticles. An ultrasonic probe was used to aid in dissolving the drug,keeping the solutions below 25° C. by cooling as needed in an ice bath.

[0170] The determination of Pd-BPheid concentration in the Cremophorsolution was performed by dilution into methanol. The absorbancespectrum was measured over 740-780 nm. The peak value was compared withthe results from a known concentration of Pd-BPheid.

[0171] (c) Additional formulations were prepared using Tween 80 andethanol to solubilize Pd-BPheid (1 mg Pd-BPheid/ml solution).

Example 7 In Vivo Toxicity Studies—Effect of Pd-Bpheid on Murine TumorModels

[0172] Two sets of experiments involving murine tumor models were usedto assess the phototoxicity of Pd-Bpheid.

[0173] (a) The photodynamic responsiveness of Pd-BPheid was firstlyevaluated in two murine tumor models: BA—mammary adenocarcinoma andradiation induced fibrosarcoma (RIF-1)

[0174] Photodynamic therapy parameters: Mice with tumors measuring 5-7mm in diameter were entered into PDT experiments. Three Pd-BPheid drugdoses (1, 5 and 10 mg/kg) and two light doses (100 and 300 Joules/sq.cm)were evaluated. A formulation of Pd-BPheid dissolved in Cremophor wasadministered by i.v. tail injection. PDT light exposure was startedeither 15 minutes, 1 hour or 4 hours following injection. Three micewere treated under each treatment condition unless initial resultsdemonstrated lethal toxicity or non-responsiveness. A titanium sapphirelaser tuned to 757 nm was used as the light source for PDT. Lasergenerated light was coupled into quartz fibers for delivery of light totumors. A light power density of 75 mW/sq.cm was used. Tumor size wasmeasured 3 days per week following PDT treatments and the percentage oftumor cures (defined as no tumor recurrence for 40 days post treatment)was determined.

[0175] In vivo PDT Response: Tables 4 and 5 hereinafter providesummaries of the PDT treatment results for C3H mice transplanted witheither the BA mammary carcinoma or the RIF-1 fibrosarcoma. Each tableindicates the following parameters: 1) intravenous drug dose expressedin mg/kg; 2) laser treatment parameters, including the total light dose(J/cm²), the wavelength (757 nm), the light dose rate (mW/cm²), and thetime interval (between treated for each group, 4) toxicity (four micedied shortly after treatment), 5) tumor regrowth (consisting of thenumber of days between PDT treatment and tumor recurrence), and 6) thenumber of mice (and percentage) with Pd-BPheid PDT induced tumor cures.

[0176] As shown herein, Pd-BPheid mediated PDT was found to induce botha classical and an efficient tumoricidal response in two mouse tumormodels. PDT mediated tumor responsiveness was directly correlated withdrug dose, light dose and time interval between drug administration andlight treatment. Specifically, higher drug doses and/or higher lightdoses produced enhanced responses. The BA mammary carcinoma was found tobe more responsive to Pd-BPheid mediated PDT than comparable PDTtreatments of the RIF-1 fibrosarcoma. Pd-BPheid mediated PDT waseffective when light treatments were initiated within 1 hour of drugadministration, and was not effective when a 4-hour interval betweendrug administration and light treatment was used.

[0177] (b) In the second set of experiments, the phototoxicity ofPd-BPheid was assessed in a mouse tumor model transplanted with HT29human colon adenocarcinoma.

[0178] Animal and tumor model: Solid tumor tissue (diameter 2 cm)removed from donor mouse immediately after death was mechanicallycrushed in 1 ml of 0.9% saline solution and the solution (0.1 ml) wasinjected s.c. into one hind leg of each mouse. Mice were included forexperiments when the tumor diameter was 8-10 mm. Tumors were grafteds.c. in 8-week aged Swiss nude mice 10 days before experiment.

[0179] Phototoxic studies: 0.15 ml Pd-BPheid was injected i.v. at 15mg/kg. Mice were anesthetized with thiopental at 40 mg/kg just beforeirradiation. At 30 min, 1 h, 4 h or 24 h after injection, mice wereirradiated with a titanium sapphire laser at 300 mW/cm², mean diameterwere measured to adjust time irradiation to obtain 200 or 300 J/cm².Control mice not injected with Pd-BPheid were also irradiated in sameconditions. The tumor growth delay induced by PDT was analyzed byequivalence with tests realized in experimental radiotherapy. For invivo studies and for each separate experiment, all results were the meanof 2 or 3 separate experiments and for each separate experiment, 2 micewere used for each experimental condition.

[0180] Concerning tumoral growth studies, results are expressed astumoral index variations with reference (=1) corresponding to tumoralindex from non-treated cells. The tumoral index was calculated asfollows:

[0181] Tumoral index=(largest tumoral diameter+perpendicularly oppositediameter)/2.

[0182] Temperature variation studies: to assure that the thermic effectwas not excessive, temperature variation was measured for the halogenlamp and the titanium sapphire laser irradiation using non-absorbingalumin-embedded microthermocouples.

[0183] The results of this experiment are the following:

[0184] (i) 763 nm Irradiation at 200 J/cm²:

[0185] A tumor growth decrease (as compared to controls) was observedfor the conditions 30 min and 4 h after injection. A decrease of tumorindex was observed up to 7 days for the conditions 1 h and 24 h afterinjection.

[0186] (ii) 763 nm Irradiation at 300 J/cm²:

[0187] A tumor growth decrease was observed (as compared to controls)for the conditions 30 min and 24 h after injection. A decrease of tumorindex was observed up to 7 days for the conditions 1 h and 4 h afterinjection.

[0188] (iii) 300 J/cm² Irradiation 1 h After Injection:

[0189] A tumor growth decrease was observed (as compared to controls)for the condition 773 nm up to 5 days and for the conditions 753 nm and763 nm up to 12 days. The maximum tumor growth decrease was observed for763 nm.

[0190] (iv) 300 J/cm² Irradiation 24 h After Injection:

[0191] A tumor growth decrease was observed (as compared to controls)for the condition 753 nm up to 4 days and for the conditions 763 nm and773 nm up to 12 days. The maximum tumor growth decrease was observed for773 nm.

[0192] No excessive temperature variation was observed during halogenlamp or titanium sapphire irradiation of mice.

[0193] In summary of this study, the optimal wavelength of irradiationwas found to be 773 nm. The delay between injection and illumination hadan influence on the tumor response. At 764 nm, a one hour delay wasshown to be the most efficient. When using a 773 nm wavelength, the mostefficient delay was 24 hours. TABLE 4 C3H/BA Mammary Carcinoma Responseto Pd-BPheid Number of Toxicity Animals with Number of (TreatmentPrimary Tumor Drug Dose Light Animals Associated Regrowth (Days Summary(mg/Kg) Parameters Treated Death) to Recurrence (cures) %  1 i.v. 300J/cm² 1 0 1 (1 da) − 757 nm no response 75 mW/cm² 15 min interval  5i.v. 300 J/cm² 3 0 + 757 nm 1 (41 days) 75 mW/cm² 2 (40 days) 15 min100% interval  5 i.v. 300 J/cm² 3 0 1 (11 days) + 757 nm 2 (41 days) 75mW/cm² 66.6% 1 hr interval 10 i.v. 300 J/cm² 3 0 + 757 nm 2 (41 days) 75mW/cm^(w) 1 (41 days) 1 hr 100% interval 10 i.v. 300 J/cm² 2 0 2 (1 day)− 757 nm no response 75 mW/cm² 4 hr interval 10 i.v. 100 j/cm² 3 0 + 757nm 2 (42 days) 75 mW/cm² 1 (41 days) 15 min 100% interval 10 i.v. 100J/cm² 3 0 1 (5 days) + 757 nm 2 (40 days) 75 mW/cm² 66.66% 1 hr interval

[0194] TABLE 5 RIF-1 Response to Pd-BPheid Number of Animals withToxicity Primary Number of (Treatment Tumor Regrowth Drug Dose LightAnimals Associated (Days to Summary (mg/Kg) Parameters Treated Death)Recurrence (cures) %  1 i.v. 300 J/cm² 2 0 2 (1 da) − 757 nm no response75 mW/cm² 15 min interval  5 i.v. 300 J/cm² 3 0 1 (5 days) + 757 nm 1(12 days) 1 (40 days) 75 mW/cm² 33.33% 15 min interval  5 i.v. 300 J/cm²3 0 1 (4 days) + 757 nm 1 (2 days) 75 mW/cm² 1 (7 days) 1 hr interval 10i.v. 300 J/cm² 3 2 + 757 nm 2 (1 day) 1 (41 days) 75 mW/cm^(w) 33.33% 15interval 10 i.v. 300 J/cm² 3 2 + 757 nm 2 (1 day) 1 (41 days) 75mW/cm^(w) 25% 1 hr interval 10 i.v. 300 J/cm² 1 0 2 (1 day) − 757 nm noresponse 75 mW/cm² 4 hr interval 10 i.v. 100 j/cm² 3 0 2 (12 days) + 757nm 1 (7 days) 75 mW/cm² 15 min interval 10 i.v. 100 J/cm² 3 0 2 (3days) + 757 nm 1 (6 days) 75 mW/cm² 1 hr interval

Example 8 Morphological Evaluation of A431 Human Epithelial CarcinoidCells after Pd-BPheid and BChl-Ser Based PDT

[0195] This experiment was performed in order to examine thetime-dependent morphological changes occurring after PDT with Pd-BPheidor BChl-SerOMe on A431 human epithelial carcinoid cells.

[0196] (i) Materials:

[0197] The Pd-BPheid was prepared as in Example 1 above and the serinemethyl ester BChl-SerOMe was prepared as in EP 584552.

[0198] (ii) Light Source:

[0199] Halogen lamp (Osram, Germany, 100 W), with 4.5 cm water filterand cut off filter >650 nm. The cells were illuminated for 10 minutes,15 mW/cm2, a total energy fluency of 9 J/cm². For illumination, theculture plates were placed on a glass table to provide the light fromthe bottom.

[0200] (iii) Phototoxicity Study:

[0201] A431 cells (5×104 cells) were seeded in 3 cm dishes in duplicatesand cultured to 75% confluency in Dulbecco's modified Eagle's medium(DMEM)+F12 (1:1), buffered with HEPES (25 mM, pH 7.4), fetal calf serum(FCS) with penicillin (0.06 mg/ml) and streptomycin (0.1 mg/ml).Pd-BPheid or BChl-Ser were added to the cells at the corresponding LD90concentration (0.1 and 1 μM, respectively). After a 4-hour period thecells were washed with culture medium and the cells were illuminatedwith the light source above. Phase contrast microscopic examination wasperformed at different time points after illumination (0, 0.5, 4 and 24hours post-PDT) using Zeiss Axiovert-35 light microscope (magnification×320) equipped with a Contax 35 mm SLR camera. In the second dish ofevery duplicate, cell viability was assessed 24 hours post-PDT usingneutral red viability assay (Zhang SZ., 1990, Cell Biol Toxicol6(2):219-234).

[0202] (iv) Results:

[0203] Both sensitizers caused significant changes in the cellmorphology. Pd-BPheid caused a fast alteration in the cells membranestructure (30 minutes), the cells rapidly shrinked and fibrousconnections were formed, connecting the cells membrane with the originalfocal adhesion points (fibrous phenotype). After 4 hours, 90% of thecells lost most qf their inner volume and a large portion of themdetached from the dish, no further change was observed after 24 hours(FIG. 4, right column). Bchl-Ser showed a different pattern of timedependant morphological changes that could be observed only after 4hours. Membrane blabbing was seen as dark vesicles budding out from thecells membrane. No significant volume decrease was observed over 24hours and after this period most of the cells were attached to the dishbut appeared hollow (blabbing phenotype, FIG. 4, Left column). Twentyfour hours after illumination, neutral red viability assay was performedwhich confirmed 90±7% cell killing in both of the experimental groups.In FIG. 4, the fibrous phenotype is represented in the right column andthe blabbing phenotype is represented in the left column. The solidwhite arrows show the formations of the fibers or the blabs.

Example 9 Photocytotoxicity of Pd-BPheid and BChl-SerOMe on the HumanBladder Carcinoma Cell Line ECV304

[0204] This experiment was carried out for assessing the photocytotoxiceffects of the photosensitizers Pd-BPheid and BChl-SerOMe on ECV304human bladder carcinoma cells.

[0205] (i) Materials:

[0206] As in Example 8(i).

[0207] (ii) Light Source:

[0208] As in Example 8(ii).

[0209] (iii) Phototoxicity Study:

[0210] ECV304 cells (2×10⁴ cells per well) were cultured in M-199, 10%FCS with penicillin (0.06 mg/ml) and streptomycin (0.1 mg/ml) in 96-wellto confluence (˜2×10⁵ cells per well). Incubation with increasingconcentrations of Pd-BPheid or BChl-SerOMe with the cells for 4 hourswas followed by washing with fresh culture medium and illumination asdescribed above Sec. 1. Twenty-four hours after illumination, cellviability was assessed using neutral red viability assay. The followingcontrols were used: Light Control: irradiated cells, not treated withsensitizer; Dark Control: non-irradiated cells, treated with sensitizerin the dark; Untreated Control: cells not treated with sensitizer andunirradiated were used for calculation of 100% survival(Rosenbach-Belkin V. et al., 1996, Photochem Photobiol 64(1) :174-181)

[0211] (iv) Results:

[0212] Both Pd-BPheid and BChl-SerOMe exhibited dose and light dependentcytotoxicity on ECV304 cells (FIG. 5). The corresponding LD₅₀ values are19 and 1000 nM. Morphological changes post-PDT were consistent with theobservations made with A431 cells (data not shown).

Example 10 PDT of Pd-BPheid and Pd-BPheid-ethyl Ester on M2R MouseMelanoma Cells

[0213] The aim of this experiment was to test the effect of Pd-BPheidand Pd-BPheid-ethyl ester on M₂R cells.

[0214] (i) Materials:

[0215] Pd-Bpheid was prepared as in Example 1 above and thePd-Bacteriopheophorbide a ethyl ester (Pd-Bpheid-ethyl ester) wasprepared as described in WO 97/19081.

[0216] (ii) Light Source:

[0217] As above in Example 8(ii) but cells were illuminated for 10minutes, 12 mW/cm², a total energy fluency of 7 J/cm².

[0218] (iii) Phototoxicity Study:

[0219] M₂R cells were cultured as monolayers in Dulbecco's modifiedEagle's medium (DMEM)+F12 (1:1), buffered with HEPES (25 mM, pH 7.4).Fetal bovine serum (FBS) (10%), glutamine (2 mM), penicillin (0.06mg/ml) and streptomycin (0.1 mg/ml) were included and the cells weregrown at 37° C. in a humidified atmosphere containing 8% CO₂. Forphototoxicity analysis cells (1×10⁴ cells/well) were cultured in 96-wellplates for 24 hours to an approximate density of 2×10⁴ cells/well.Pigments were dissolved directly in culture medium or in ethanol 95% andfurther diluted in culture medium to a final concentration of 1%ethanol. The diluted pigments were added and the cells were incubated inthe dark for four hours at 37° C. Prior to illumination, the cells werewashed once and replaced with fresh culture medium. The plates were thenilluminated from the bottom for 10 minutes at room temperature andplaced in the culture incubator at 37° C. in the dark. Cell survival wasdetermined 24 hours later. The following control systems were used: DarkControl: untreated cells kept in the dark; Light Control: cells nottreated with sensitizer that were illuminated; Dark Toxicity: cellstreated with pigment but kept in the dark. Cell survival was determinedby [³H]-thymidine incorporation as described earlier (WO 97/19081).

[0220] (iv) Results:

[0221] As can be seen in FIG. 6A, when the pigments were dissolved inethanol 95%, Pd-BPheid had a LD₅₀ of 0.03 μM, while the Pd-BPheid-ethylester had a LD₅₀ of 0.07 μM. When the pigments were dissolved directlyin culture medium containing 10% serum, only the Pd-BPheid was fullyactive while the Pd-BPheid-ethyl ester was not active at all up to 1 μM,the highest concentration tested (FIG. 6B).

Example 11 PDT of Pd-BPheid on M2R Mouse Melanoma and Human HT29 ColonCarcinoma Cells

[0222] These experiments were aimed at determining the phototoxic effectof Pd-BPheid toward two cell lines: M2R mouse melanoma and human HT29colon carcinoma cells.

[0223] (i) Materials:

[0224] Pd-Bpheid was prepared as in Example 1 above.

[0225] (ii) Light Source:

[0226] The light source was a Xenon fluorine LS3-PDT lamp (Bio-Spec,Russia), with 10 cm water filter and 720-850 nm light band. The cellswere illuminated for 10 minutes, 12 mW/cm², at a total energy of 7J/cm².

[0227] (iii) Phototoxicity Study:

[0228] Analysis was performed with the same protocol as described above(Example 10) with the following changes: Pd-BPheid was dissolveddirectly in medium containing 10% serum and then added to the cells.Survival of M2R cells was determined by [³H]-thymidine incorporation andthat of human HT29 cells with the MTT assay (Merlin J L et al., 1992 EurJ Cancer 28A:1452-1458).

[0229] (iv) Results:

[0230] As can be seen in FIG. 7, human colon HT-29 cells show lowersensitivity toward this pigment (LD₅₀ of 0.5 μM), while the M₂R cellswere about 10 times more sensitive (LD₅₀ of 0.03 μM).

Example 12 In Vivo PDT of M2R Mouse Melanoma Tumors with Pd-BPheid

[0231] The aim of this experiment was to study PDT of M2R mouse melanomatumors in CD1 nude mice with 2.5 mg/Kg Pd-Bpheid.

[0232] (i) Materials:

[0233] P d-Bpheid was prepared as in Example 1 above.

[0234] (ii) Mice:

[0235] C D1 nude mice (25-30 g)

[0236] (iii) Anesthesia:

[0237] i.p injection of 50 μl of Ketamine/Rumpon (vol/vol=85/15).

[0238] (iv) Tumor Implantation:

[0239] Mice were implanted with 106 M2R cells on the back and tumorsarose to the treatment size (7-8 mm) within 2-3 weeks.

[0240] (v) Light Source:

[0241] Osram150 W halogen photo-optic lamp 64643 (D. K. Keller et al1999, Int J Hyperthermia 15:467-474) equipped with λ=650-900 mn spectralwindow, 300 mW/cm-2. Illumination was for 30 min.

[0242] (vi) PDT Protocol:

[0243] The anesthetized mouse was i.v injected with the pigment and thetumor immediately illuminated. At the end of treatment the mouse wasplaced back in the cage. Photographs of the tumor were taken before andat the times indicated.

[0244] Experiment 1

[0245] Preparation of Sensitizer:

[0246] Two mg Pd-BPheid were dissolved in 0.25 ml Cremophor EL followedby 20 min sonication. 0.075 ml 1,2-propylene glycol were added andsonication was continued for another 15 min. Then 0.9 ml of 0.15 mM NaClwere added followed by 5 min sonication. The sample was centrifuged for12 min at 13,000 rpm (Eppendorf). The final calculated concentration ofPd-BPheid based on spectrum in chloroform was 0.5 mg/ml.

[0247] PDT of Tumor:

[0248] Pd-BPheid 2.5 mg/kg was i.v injected to CD1-Nude mouse bearingM2R melanoma tumor. The tumor was illuminated for 30 min at 300 mW cm⁻².The temperature of the mouse skin tumor area was 37.7-38° C. Theresponse of tumor was followed 1 and 4 days after treatment. The resultsare shown in FIG. 8.

[0249] Experiment 2

[0250] Preparation of Sensitizer:

[0251] Two mg Pd-BPheid were dissolved in 0.1 ml methanol, 0.1 ml 0.1 MKH₂ PO₄, pH=8.0 and 0.9 ml PBS and sonicated for 10 min. The methanolwas evaporated with Argon and 20% of Cremophor EL: 1,2-propylene glycol(3:1) was added following by 15 min sonication. The sample wascentrifuged for 8 min on 13,000 rpm the final calculated concentrationof Pd-BPheid based on spectrum in chloroform was 0.5 mg/ml.

[0252] PDT of Tumor:

[0253] Pd-BPheid 2.5 mg/kg (120 μl) was i.v administered to CD1-Nudemice bearing M2R melanoma tumor. The tumor tissue was illuminated for 30min at 300 mW cm⁻². The temperature of the mouse skin tumor area was37.7-38° C. The response of tumor was followed 1 and 4 days aftertreatment. The results are shown in FIG. 9.

[0254] Results

[0255] As shown in FIGS. 8 and 9, PDT of M2R melanoma tumors with 2.5mg/Kg Pd-Bpheid as described above induces severe inflammatory responsewith necrosis of the tumor within 24 h.

Example 13 Pd-BPheid Based PDT Reduces Rate of C6 Glioma MetastasisFormation in Mice: Advantage over Surgery

[0256] These experiments were conducted in order to compare thetherapeutic potential of Pd-BPheid and BChl-SerOMe based PDT, and theprobability of metastasis spread by Pd-BPheid and BChl-SerOMe based PDT.

[0257] (i) Materials:

[0258] Pd-BPheid (prepared as in Example 1) or Pd-BPpheid-SerOMe 5 mg/kgin 20% Cremophor EL.

[0259] (ii) Light Source:

[0260] The light source was a Xenon fluorine LS3-PDT lamp (Bio-Spec,Russia), with 10 cm water filter and 720-850 nm light band.

[0261] (iii) Mice:

[0262] CD1 nude mice.

[0263] (iv) Tumors:

[0264] Mice were implanted with 10⁶ C6 glioma cells in the foot of thehind leg. Tumors were treated when reached a length of 7-8 mm.

[0265] (v) Anesthesia:

[0266] 50 μl of Vetalar/Rumpon (vol/vol=85/15).

[0267] (vi) Analgesia:

[0268] Oxycodone (12 mg/liter) added in 5% sucrose drinking water, as oftreatment (amputation or PDT) for one week.

[0269] (vii) Protocol:

[0270] Three groups (10 mice in each) were i.v. injected with 5 mg/Kg ofsensitizer (Pd-BPheid or Pd-BPpheid-SerOMe) and immediately illuminatedat 200 mw/cm², for 30 minutes, and the animals were allowed to recoverin the cage.

[0271] Groups 1 and 2:

[0272] Animals which received PDT Pd-Bpheid and Pd-BPpheid-SerOMe,respectively. Tumor response and metastasis formation in groin werefollowed for 4 weeks.

[0273] Group 3:

[0274] Animals which were amputated at the ankle joint (paired withgroup 1) and metastasis formation in groin was followed for 4 weeks. Theparameters of response to PDT were the percent of animals with tumornecrosis and disappearance, out of the total number of treated animals.Metatstasis was manifested by appearance of tumors in the groin orelsewhere. The endpoints considered were: follow up for 4 weeks,spontaneous death, tumors reached a diameter of 2 cm, metastasis,whichever came first.

[0275] (viii) Results:

[0276] The results of tumor flattening (disappearance) are shown on FIG.10. While on day 11 the response to Pd-BPheid was stronger than toPd-BPheid-SerOMe (100% and 80% tumor flattening, respectively), later,on day 28, the percent of response was similar, about 60%. The declinein tumor flattening in the long term is due to some tumor re-growth insome of the treated animals, probably due to mismatch of light field andtumor area.

[0277] The results of metastasis appearance are shown in FIG. 11. Thesurgical treatment by leg amputation yielded a substantially higherpercent of metastasis in comparison to PDT (up to 78%). In addition, themetastasis after amputation appeared much earlier. The frequency ofmetastasis after PDT with Pd-BPheid was the lowest (up to 23%). Thisresult is similar to that obtained with Pd-BPheid-SerOMe and the mainadvantage of Pd-BPheid is delay of metastasis appearance. PDT withPd-BPheid or Pd-BPheid-SerOMe are curative for C6 glioma tumors.Metastasis formation after PDT is substantially lower when compared withsurgical treatment.

What is claimed is:
 1. A method for tumor diagnosis which comprises: (a)administering to a subject suspected of having a tumor, a compound ofthe formula I, I′ or I″

wherein A represents OH, OR₁, —O—(CH₂)_(n)—Y, —S—(CH₂)_(n)—Y,—NH—(CH₂)_(n)—Y, —O—(CH₂)₂—NH₂, —O—(CH₂)₂—OH, —NH—(CH₂)₂—NH—BOC or—N—(CH₂—CH═CH₂)₂ wherein R₁ represents Na⁺, K⁺, (Ca²⁺)_(0.5),(Mg²⁺)_(0.5), Li⁺, NH₄ ⁺ ^(+NH) ₃—C (CH₂OH)₃, ⁺NH₃—CH₂—(CHOH)₄—CH₂OH,⁺NH₂(CH₃)—CH₂—(CHOH)₄—CH₂OH or ⁺N (C_(n),H_(2n),₊₁)₄; R₂ represents H,OH or COOR₄, wherein R₄ is C₁-C₁₂ alkyl or C₃-C₁₂ cycloalkyl; R₃represents H, OH or C₁-C₁₂ alkyl or alkoxy; n is 1, 2, 3, 4, 5 or 6, Yis —NR′₁R′₂ or —⁺NR′₁R′₂R′₃, X⁻ wherein R′₁, R′₂ and R′₃ independentlyfrom each other represent —CH₃ or —C₂H₅; X is F, Cl, Br or I, n′ is 1,2, 3 or 4, and wherein ★ denotes an asymmetric carbon and—represents asingle saturated bond or a double unsaturated bond; and (b) irradiatingthe subject by standard procedures and measuring the fluorescence of thesuspected area, wherein a higher fluorescence indicates tumor sites. 2.A method according to claim 1 which comprises administering a compound Iof the formula and optical configuration as indicated below:

wherein A is OH or OR₁, and R₁ is as defined in claim
 1. 3. The methodaccording to claim 2 which comprises administering the compound whereinA is OH, herein identified as Pd-Bacteriopheophorbide a (Pd-BPheid).